M-current is abundant in the nervous system. This potassium current is known to play an important role in the control of cell excitability and action potential firing through its unique voltage-dependent properties. Receptor-mediated modulation of M-current has dramatic effects upon neuronal excitability. Its suppression causes membrane depolarization and an increase in cell firing, while enhancement stabilizes cell excitability. The amplitude of the M-current is also influenced by the intracellular calcium concentration, with modest levels enhancing the current and larger loads causing inhibition. Recent findings have placed the M-current in a unique position. It is blocked in hippocampal pyramidal neurons by compound known to enhance cognition and may underlle seizure-generation activity in cortical cells. Therefore, it is imperative to understand the mechanisms that control the amplitude of the M-current and how its suppression by receptor activation occurs. The M- channel resides in two dominant gating states, consisting of either high (mode 2) or low (mode 1) open probability activity. The characteristically slow macroscopic current relaxations arise principally from mode 2 channel activity. First, this project will resolve the identity and role of the endogenous enzymes that control M-channel modal gating and set the amplitude of the current. Secondly, the second messenger that couples receptor activation to M-current suppression will be identified. M-channel modal gating is regulated by phosphorylation. Endogenous kinase and phosphatase enzymes remain associated with the patch upon excision. Activation of the kinase by a modest increase in intracellular calcium promotes mode 2 openings while phosphatase activity produces mode 1 activity, an effect mimicked by the calcium-dependent phosphatase, calcineurin. This application will determine the identity of the endogenous enzymes and examine their calcium dependence, to resolve whether the effect of intracellular calcium on the macroscopic M-current can be explained by the relative activities of associated calcium dependent kinase and phosphatase. Activation of calcineurin is not the messenger coupling receptor activation to the suppression of M-current. The kinetic consequence of receptor modulation is a selective reduction in mode 2 M-channel behavior, an effect different from that of calcineurin. The proposal is also designed to understand how mode 2 M- channel activity is sustained in inside-out patches. Once achieved, putative messengers will be applied to excised patches to determine which can truly mimic the effect of receptor activation. This application will identify those intracellular messengers that regulate this current, enabling a better understanding of the role of M-current modulation in neuronal excitability.